Sonic hedgehog (Shh) acts as a mitogen and cell survival factor in many adult processes during normal tissue renewal and in many types of cancer, but acts as a morphogen in several developmental contexts. How the mitogenic role of Shh is integrated with the morphogenetic role in developmental contexts is still poorly understood. In the limb, Shh regulates both digit number and identity of different digits (A-to-P,thumb to pinky). Shh is thought to act as a morphogen forming a gradient along the limb AP axis, with higher concentrations specifying more posterior digit types. We have determined the time-requirements for Shh function in limb (using a tamoxifen-regulated Cre to remove Shh at different times in mice). To perform this analysis, we generated and characterized a novel conditional Cre recombinase line selectively expressed in early limb mesoderm, neural crest, gut and tailbud. This line provides an excellent tool available to the scientific community to illuminate different temporal roles of key developmental regulators in several important developmental models using mouse mutants, as well as for genetic lineage tracing studies in mice. Our results, deleting Shh function at different time points, are most consistent with a model in which Shh activity is required only very transiently (several hours) to specify the complete complement of 5 different digit types, but is required for a prolonged time (about 2 days) to maintain cell survival and proliferation, enabling 5 normal digits to form. To further test this model for Shh function, we have assessed whether restoring cell survival can rescue normal digit formation in mutant embryos after a transient period of Shh activity that is terminated by Shh gene deletion. To rescue cell survival, the compound mutant for the pro-apoptotic Bcl2 family members Bax/Bak (which play roles in normal interdigital apoptosis) has been introduced to inactivate the intrinsic death pathway. Our preliminary results indicate that both normal digit number and pattern (morphogenesis) can be rescued by simply restoring cell survival and proliferation in Shh mutant embryos. In addition, genetic lineage tracing of cells in the limb that have responded to Shh signals at different time points indicates that Shh only signals directly to the very posterior part of the limb bud at the time when all 5 digit progenitors have been specified. This result implies that Shh acts indirectly in the early limb bud, via a system of relay signals, to specify digits. Genetic manipulations to artificially enforce Shh-response in the posterior limb bud in embryos lacking all Shh function (Shh KO) partly rescues anterior digit formation, and strongly suggests the presence of relay signals acting downstream of Shh. Our results thus far challenge the view that Shh behaves as a classic morphogen and suggest that Shh acts initially by generating distinct limb bud zones. Furthermore, our results indicate that there are 2 classes of Shh responsive target genes, those that respond to a transient signal and become stably expressed, and those that require continuous signaling to maintain expression. We are comparing the transcriptomes of Shh mutant and rescued limb buds to characterize the types of genes in these two differentially regulated target classes. In parallel, single cell transcriptome analysis from normal limb buds will be performed to identify expression signatures in the transient Shh signaling phase and characterize immediate-early response zones. Understanding the proliferative and anti-apoptotic actions of Shh in the context of these differentially regulated target classes, will provide a reference for deciphering and intercepting Shh roles in cancer.